Riboswitches are RNA sensors located in untranslated regions of mRNA that regulate expression of adjacent genes in bacteria and some eukaryotes. In many cases, riboswitches act through a feedback mechanism by binding target small molecule ligands that are critical elements in important cellular processes. A recently discovered class of riboswitches, termed GEMM, was found upstream of genes that influence membranes, motility, or the extracellular environment. Predicted secondary structural elements of the RNA include two highly conserved stems whose internal loops are thought to confer ligand binding specificity. Experiments have demonstrated that cyclic diguanosine monophosphate (c-diGMP), an important second messenger molecule, binds to the GEMM riboswitch with high affinity and selectivity. Cellular functions such as motility and pathogenesis were previously associated with fluctuations in c-diGMP concentration, although the molecular basis for this observation is presently unknown. The goal of this work is to explore the basis of c-diGMP ligand binding and gene regulation by the GEMM riboswitch using both biochemical and structural analysis. The specific contacts to the RNA molecule required-for binding of c-diGMP will be established by mutagenesis (aim 1) and the dynamics of ligand binding probed by real-time NMR (aim 2). This work will complement current efforts toward an x-ray crystal structure and will help to reveal structural changes undertaken by the RNA molecule upon ligand binding. Modulation of gene expression by c-diGMP in both wild-type and hybrid GEMM riboswitch structures in a cellular context will be tested by monitoring the electron transfer ability of the bacteria Geobacteria sulfurreducens (aim 3). PUBLIC HEALTH RELEVANCE: GEMM riboswitches are gene control elements found to influence gene expression in a variety of organisms, including several pathogenic bacteria. These elements are responsive to fluctuations in the second messenger molecule cyclic diGMP and act to modulate expression of genes important for cell motility and virulence, among others. Understanding the molecular basis by which gene control is accomplished, through both structural and biochemical analysis, may help to establish the GEMM riboswitch as a potential drug target.